Small compounds targeting tacc3

ABSTRACT

The present invention provides a small compound targeting at TACC3. The present invention further provides a drug, particularly, an anticancer agent, comprising the small compound targeting at TACC3. A compound represented by the general formula (I) or a pharmaceutically acceptable salt, solvate, or ester derivative thereof binds to TACC3 and inhibits cell growth. Thus, these compounds can be used as drugs, particularly, anticancer agents.

TECHNICAL FIELD

The present invention relates to a small compound targeting at TACC3.The present invention also relates to a drug, particularly, ananticancer agent, based on the small compound targeting at TACC3.

BACKGROUND ART

Microtubules constitute spindles formed during cell division. Sincetumor cells are in active cell division, agents inhibiting cell divisionare effective as anticancer agents. For this reason, anticancer agentstargeting microtubules as the main structure of spindles or tubulin as aconstituent protein thereof have been classically developed and used intreatment regardless of solid tumor or hematological tumor.

In fact, many compounds, such as vincristine and paclitaxel, whichtarget microtubules, are widely used as anticancer agents. These agentsare considered to exert their anticancer effects as a result ofinhibiting spindle formation in tumor cells and thereby suppressing celldivision.

These compounds, however, are known to cause serious adverse reactionssuch as peripheral neuropathy, because they target not only microtubulesin spindles but microtubules in normal cells (e.g., Patent Literature 1and Non Patent Literature 1). Also, particular isoforms of tubulin havebeen reported to have resistance to anticancer agents. It has beentherefore required to develop a novel drug selectively targetingmicrotubules in tumor cells (Non Patent Literature 2).

In search for a compound selectively targeting microtubules in tumorcells, the present inventors have conducted analysis by focusing onTACC3, which are reportedly involved in microtubular polymerization andabnormally expressed in various tumors.

A gene encoding the TACC3 (transforming acidic coiled-coil 3) protein isconsidered to be a so-called cancer gene (e.g., Non Patent Literatures 3to 8 and Patent Literature 2). TACC3 is also known to participate inspindle formation or the control of a mitotic apparatus involved inchromosome partitioning and cell division (e.g., Non Patent Literature9). In addition, TACC3 and TOGp (tumor over-expressed gene) known tointeract with TACC3 are overexpressed in various cancers.

The suppression of TACC3 expression induces different phenomenadepending on the cell line used. For example, reduction in microtubularpolymerization (Non Patent Literature 10) and apoptosis induced bychromosomal imbalance (Non Patent Literature 9) are reportedly observed.

These phenomena are explained, on the basis of findings obtained inXenopus or Drosophila, by models in which TACC3 binds to TOGp andstabilizes microtubular polymerization in centrosomes during mitosis,thereby controlling cell division (Non Patent Literature 11).

The present inventors have experimented the suppression of TACC3expression using TACC3-conditional knockout mice. As a result, thepresent inventors have found that lymphoma undergoes regression due toapoptosis, whereas normal thymus cells are found to express TACC3, butare not evidently affected thereby (Non Patent Literature 12). Thus, thepresent inventors have considered that compounds targeting at TACC3 mayserve as excellent anticancer agents selectively acting on tumor cells,and already disclosed a method for screening for an anticancer agenttargeting at TACC3 (Patent Literature 3).

CITATION LIST Patent Literature

-   Patent Literature 1: Japanese Patent Laid-Open No. 2009-167163-   Patent Literature 2: International Publication No. WO 2005/071419-   Patent Literature 3: Japanese Patent Laid-Open No. 2012-5479 Non    Patent Literature-   Non Patent Literature 1: Kavallaris, M., Nat. Rev. Cancer (2010),    10, 194-204-   Non Patent Literature 2: Sudakin, V. and Yen, T. J., BioDrugs    (2007), 21, 225-33-   Non Patent Literature 3: Charrasse, S. et al., Eur. J. Biochem.    (1995), 234 (2), 406-413-   Non Patent Literature 4: Kiemeney, L. A. et al., Nat. Genet. (2010),    42 (5), 415-419-   Non Patent Literature 5: Peters, D. G. et al., Cancer Epidemiol.    Biomarkers Prev. (2005), 14 (7), 1717-1723-   Non Patent Literature 6: Ma, X. J. et al., Proc. Natl. Acad. Sci.    USA (2003), 100 (10), 5974-5979-   Non Patent Literature 7: Ulisse, S. et al., Endocr. Relat. Cancer    (2007), 14 (3), 827-837-   Non Patent Literature 8: Still, I. H. et al., Genomics (1999), 58    (2), 165-170-   Non Patent Literature 9: Schneider, L. et al. Oncogene (2008), 27    (1), 116-125-   Non Patent Literature 10: Gergely, F. et al. Genes Dev. (2003), 17,    336-341-   Non Patent Literature 11: Peset, I., and Vernons, I. Trends Cell    Biol. (2008), 18 (8), 379-388-   Non Patent Literature 12: Yao, R. et al., Oncogene (2012), 31,    135-148-   Non Patent Literature 13: Miyazaki, I. et al., Methods Mol.    Biol. (2010) 669, 95-107-   Non Patent Literature 14: Dubovic I. P. et al., Chemist. Natural    Compounds, (2004) 40, 434-443-   Non Patent Literature 15: Yamori, T., Cancer Chemother. Pharmacol.,    (2007), 52 Suppl. 1, S74-79

SUMMARY OF INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a small compoundtargeting at TACC3. Another object of the present invention is toprovide a drug, particularly, an anticancer agent, comprising the smallcompound targeting at TACC3 and a method for producing the same.

Means for Solving the Problems

The anticancer agent of the present invention comprises a compoundrepresented by the general formula (I) given below or a pharmaceuticallyacceptable salt, solvate, or ester derivative thereof as an activeingredient.

wherein R¹ represents

OAc, NEt₂, NMe(CH₂CH₂OH), NH(CH₂CH₂NMe₂), NEt(CH₂CH₂NMe₂),N(CH₂CH₂OMe)₂, N⁺O⁻(CH₂CH₂OMe)₂, NMe(CH₂)₃Me, or N(CH₂CH₂Me)₂; R²represents Ac or H; and R³ represents H, Cl, F, or Br.

The present inventors have revealed, by experiments using mice orcultured cells, that the above-mentioned compound having a dicoumarinstructure suppresses cell division via TACC3 and exhibits an antitumoreffect. Thus, effective anticancer agents can be developed by usingthese compounds as active ingredients for the anticancer agents.Anticancer agents selectively acting on tumor cells can be produced byusing these compound in the treatment of a cancer expressing TACC3 andpreparing these compounds into anticancer agent compositions.

The anticancer agent of the present invention comprises a compound ofthe general formula (I) represented by the following formula:

or a pharmaceutically acceptable salt, solvate, or ester derivativethereof as an active ingredient.

The compounds shown above are novel compounds that have been synthesizedby the present inventors. These compounds suppress cell division inexperiments using cultured cells and as such, can be used as anticanceragents.

In the anticancer agent of the present invention, the targeted cancer isa cancer expressing at TACC3, particularly, colon cancer, ovary cancer,uterine cancer, breast cancer, esophagus cancer, lymphoma, glioma,prostate cancer, kidney cancer, or melanoma.

The compound of the present invention functions via TACC3 or aTACC3-TOGp complex and therefore acts on tumor cells expressing TACC3.Particularly, colon cancer, ovary cancer, uterine cancer, breast cancer,esophagus cancer, or lymphoma often overexpresses TACC3. An anticanceragent comprising the compound of the present invention as an activeingredient is therefore confirmed to effectively act on these cancertypes.

Furthermore, the compound of the present invention exhibits a high celldivision inhibitory or arresting effect on ovary cancer, colon cancer,glioma, prostate cancer, kidney cancer, or melanoma. An anticancer agentcomprising the compound of the present invention as an active ingredientis therefore confirmed to effectively act on these cancer types.

In addition, mouse experiments on lymphoma have revealed anapoptosis-induced tumor regression effect through the inhibition ofTACC3 and also revealed the cell death-inducing effect of the compoundof the present invention. An anticancer agent comprising the compound ofthe present invention as an active ingredient is therefore confirmed toeffectively act on these cancer types.

The anticancer agent composition of the present invention is acomposition for oral administration.

Since the compound of the present invention has been found effectivethrough oral administration in experiments using mice, the compositioncan be provided as an oral formulation. Such oral administrationeliminates the need of injection by physicians, and patients cancontinue treatment even at home without visiting hospitals. In addition,such oral formulations can be relatively easily used in combination withother agents and as such, can be expected to have a wide range ofapplications and effects.

The present invention also provides a method for producing an anticanceragent composition, comprising mixing the compound of the presentinvention or a pharmaceutically acceptable salt, solvate, or esterderivative thereof with a pharmaceutically acceptable excipient.

The compound disclosed in the present invention can be mixed as anactive ingredient with an excipient to thereby produce an anticanceragent composition selectively acting on tumor cells expressing TACC3.

Advantageous Effects of Invention

The present invention can provide a drug, particularly, an anticanceragent, targeting at TACC3. Also, the compound disclosed in the presentinvention selectively inhibits the functions of spindles and mitoticapparatuses via a TACC3-TOGp complex and therefore selectively targetsonly actively dividing tumor cells expressing TACC3. The compound of thepresent invention is orally administrable and as such, can be expectedto be widely applied clinically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic diagram of a method for screening for a compoundbinding to TACC3 using a chemical array.

FIG. 2 Diagram showing a dose-dependent mitotic arrest by an NPDcompound.

FIG. 3 Diagram showing a dose-dependent mitotic arrest by SPL(Spindlactone).

FIGS. 4A and 4B Diagrams showing results of analyzing cell fate usingSPL-treated SKOV-3 cells and OVCAR-3 cells.

FIGS. 5A and 5B Diagrams showing the percentage of spindle morphology ofcells containing abnormal spindles induced by SPL treatment. FIG. 5A isa diagram showing the results of analysis using SKOV-3 cells. FIG. 5B isa diagram showing the results of analysis using OVCAR-3 cells.

FIG. 6 Diagram showing the interaction between a TACC3-TOGp complex andSPL.

FIG. 7 Diagram showing the in vivo antitumor effect of SPL-B.

FIG. 8 Diagram showing the effect of SPL-B administration on tumors byimmunohistochemical analysis. Tumor sections were immunohistologicallystained using anti-p53, anti-p21, anti-H3K9me3, and anti-PCNAantibodies, and the percentage of positive cells is shown.

FIG. 9 Diagram showing results of analyzing the cell cycles of variouscancer cell lines using after SPL-B treatment.

FIGS. 10A and 10B Diagrams showing results of analyzing the growthinhibitory effects of SPL-A and other anticancer agents on normal cellsand cancer cells. FIG. 10A shows results of analyzing the growthinhibitory effects of SPL-A. FIG. 10B shows results of other anticanceragents.

FIGS. 11A and 11B Diagrams showing effect on cancer cell growth by thecombined use of SPL-B and paclitaxel. FIG. 11A shows a growth inhibitoryeffect by the combined use of SPL-B and paclitaxel. FIG. 11B showseffect by the single use of SPL-B.

DESCRIPTION OF THE EMBODIMENTS

The present invention provides a compound targeting at TACC3. Thecompound targeting at TACC3 means a compound that binds to the TACC3protein and thereby inhibits its effects.

TACC3 is a protein member of the TACC family. The nucleotide sequence ofa gene encoding TACC3 and the amino acid sequence thereof are disclosedon databases under, for example, GenBank Accession No. NM_(—)006342.1and UniProt Accession No. Q9Y6A5.

The disease to which the inhibition of functions of the TACC3 gene orthe TACC3 protein is advantageous is not limited and is preferably atumor. In the present invention, the terms “cancer” and “tumor” are usedinterchangeably.

TACC3 is highly expressed in tumor cells compared with correspondingnon-tumor cells. The expression means gene and/or protein expression,unless otherwise specified.

The tumor is not limited and is selected from the group consisting ofsarcoma, leukemia, biliary tract cancer, breast cancer, uterine cancer,colorectal cancer, throat cancer, esophagus cancer, stomach cancer,colon cancer, tonsillar cancer, tongue cancer, neck cancer, lymphoma,lung cancer, thyroid gland cancer, ovary cancer, kidney cancer, pancreascancer, brain tumor, myeloma, glioma, melanoma, liver cancer, prostatecancer, and urinary bladder cancer, preferably ovary cancer, breastcancer, uterine cancer, esophagus cancer, stomach cancer, colon cancer,pancreas cancer, prostate cancer, lymphoma, myeloma, glioma, kidneycancer, and melanoma, particularly preferably colon cancer, ovarycancer, uterine cancer, breast cancer, esophagus cancer, lymphoma,glioma, prostate cancer, kidney cancer, and melanoma.

The drug of the present invention is a drug for the treatment ofactively dividing cells or tumor cells expressing TACC3. The treatmentmeans the inhibition of abnormal cell growth of TACC3-expressing cells,or the induction of cell death, preferably tumor cells, highlyexpressing TACC3, thereby causing the delay or inhibition of tumorgrowth and the regression of a disease or a disorder, particularly, atumor in a subject.

Examples of the pharmaceutically acceptable salt of the compound or thedrug of the present invention include salts with inorganic and organicacids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuricacid, phosphoric acid, citric acid, formic acid, maleic acid, aceticacid, succinic acid, tartaric acid, methanesulfonic acid, andp-toluenesulfonic acid. Other examples thereof include base salts suchas sodium hydroxide, potassium hydroxide, potassium carbonate, sodiumbicarbonate, ammonia, amine salts, and trialkylamine salts.

Examples of the pharmaceutically acceptable ester derivative includeester compounds with alcohols or carboxylic acids each having 1 to 10carbon atoms, preferably methyl alcohol, ethyl alcohol, acetic acid, orpropionic acid.

Preferred examples of the pharmaceutically acceptable solvate includesolvates with water.

Such a salt, ester derivative, and solvate can be formed by thoseskilled in the art using standard techniques.

The drug of the present invention may be orally administered in thedosage form of, for example, tablets, coated tablets, sugar-coatedtablets, hard or soft gelatin capsules, solutions, emulsions, orsuspensions. Alternatively, the drug of the present invention may beintrarectally administered by use of, for example, a suppository.Alternatively, the drug of the present invention may be administeredlocally or percutaneously by use of, for example, an ointment, a cream,a gel, or a solution. Also, the drug of the present invention may beadministered parenterally, for example, intravenously, intramuscularly,subcutaneously, intraspinally, or intracutaneously by injection.Intravenous, intramuscular, or oral administration is preferred. Oraladministration is most preferred. The drug of the present invention canbe administered once or several times a day, though the number of dosesis not limited thereto.

The drug of the present invention may be mixed with a pharmaceuticallyinert inorganic or organic excipient. Examples of appropriate excipientsfor tablets, sugar-coated tablets, or hard gelatin capsules includelactose, corn starch and derivatives thereof, talc, and stearic acid andsalts thereof. Examples of appropriate excipients for use in softgelatin capsules include plant oils, waxes, fats, and semisolid orliquid polyols. Examples of excipients for preparing solutions andsyrups include water, polyols, saccharose, invert sugars, and glucose.Examples of excipients for injections include water, alcohols, polyols,glycerin, and plant oils. Examples of excipients for suppositories andlocal or percutaneous application include natural or hydrogenated oils,waxes, fats, and semisolid or liquid polyols. The drug of the presentinvention may further contain an antiseptic, a solubilizer, astabilizer, a wetting agent, an emulsifier, a sweetener, a colorant, aflavoring agent, a salt changing osmotic pressure, a buffer, a coatingagent, or an antioxidant, etc. The drug of the present invention mayfurther contain an additional therapeutically useful agent.

The effective amount or dose of the compound of the present invention isnot particularly limited and can be appropriately selected according tomethod of administration, age, body weight, and symptoms.

The subject to be treated using the drug of the present invention is amammal. The mammal can be any mammal such as mice, rats, rabbits, guineapigs, dogs, cats, cattle, horses, goats, sheep, monkeys, and humans. Themammal is preferably a companion animal such as a dog or a cat, or ahuman, more preferably a human.

Hereinafter, the present invention will be described in detail withreference to Examples. However, the present invention is not intended tobe limited by these Examples.

Example 1 Screening for Compound Binding to TACC3-DsRed Fusion Protein

The present inventors screened for compounds targeting at TACC3 using achemical array (FIG. 1).

According to the protocol of Miyazaki et al. (Non Patent Literature 13),a cell lysate containing a TACC3-DsRed fusion protein was contacted withthe chemical array to screen for a compound binding to the TACC3-DsRedfusion protein (FIG. 1, upper diagram).

The cell lysate containing a TACC3-DsRed fusion protein is prepared asfollows.

Human TACC3 cDNA was cloned into pDsRed-Express-N1 (Registered TradeMark) (manufactured by Clontech Laboratories, Inc.), which was thentransferred to HEK293T cells. Forty eight hours after transfection, thecells were collected. The obtained cells were sonicated in PBS andcentrifuged for removal of insoluble substances to obtain a cell lysate.The cell lysate was confirmed the presence of the TACC3-DsRed fusionprotein using anti-TACC3 and anti-DsRed antibodies.

The cell lysate containing a TACC3-DsRed fusion protein was contactedwith the chemical array. After washing, the TACC3-DsRed fusion proteinbound with a compound immobilized on the chemical array was detected.The detection of the TACC3-DsRed fusion protein with a compound wascarried out by the excitation of a chemical array slide with awavelength of 532 nm and the detection of fluorescence at 575 nm

As a result of screening 6800 compounds, 70 compounds were bound withthe TACC3-DsRed fusion protein. These 70 compounds recognized to bind tothe TACC3-DsRed fusion protein were each added into a culture medium ofcultured cells. Compounds inducing abnormal cell division were selectedby microscopic observation. As a result, 4 compounds were screened foras compounds inhibiting cell division (FIG. 1, lower diagram, visualscreening).

Four compounds represented by the formulas (II) to (V) given below arecompounds that bound to the TACC3-DsRed fusion protein and inducedabnormal cell division. These compounds (II) to (V) immobilized on thechemical array were registered in the Riken natural products depositorybank “RIKEN NPDepo” (http://npd.rikenjp/npd/) under NPD3574, NPD2568,NPD4089, and NPD2448 and are available from the bank.

The 4 compounds NPD3574, NPD2568, NPD4089, and NPD2448 obtained byscreening using the chemical array each have a dicoumarin structurerepresented by the formula (I) given below wherein an OH group islocated at position C₇. Thus, the structure of the formula (I) isimportant for binding to TACC3.

Example 2 Mitotic Arrest Induced by NPD Compounds

The 4 NPD compounds selected by screening using the chemical array wereanalyzed for their activity against cell division using synchronizedculture.

The 4 compounds each have a dicoumarin structure having an OH group atposition C₇, suggesting the importance of this OH group at position C₇.Thus, C₇—O— propylated NPD3574 was synthesized by propylation atposition C₇ and analyzed, together with the 4 NPD compounds, for itsinfluence on cell division of cultured cells.

Hereinafter, analysis methods will be described in detail.

(1) Preparation of EGFP-α-Tubulin-Expressing Cell

The EGFP-α-tubulin-expressing cells used in the assay will be described.In order to observe mitosis under a fluorescence microscope, cellsexpressing EGFP-α-tubulin (fusion protein of a fluorescent protein,EGFP, and α-tubulin) were prepared. A system observable under afluorescence microscope without cell fixation was prepared.

Ovary cancer cells SKOV-3 were cultured in an RPMI1640 mediumsupplemented with 5% fetal bovine serum. Stable cells expressingEGFP-α-tubulin were transduced by lentiviral transduction usingpLenti4/V5-DEST vectors (Registered Trade Mark) (manufactured byInvitrogen Corp.) and selected by the FACS sorting of infected cellsusing Zeocin (Registered Trade Mark) and subsequent FACSAria (RegisteredTrade Mark) (Becton, Dickinson and Company).

(2) Analysis of Mitotic Arrest by NPD Compound

The compounds NPD2448, NPD2568, NPD3574, NPD4089, and C₇—O-propylatedNPD3574 were each added at varying concentrations to the culture mediumof SKOV-3 cells expressing EGFP-α-tubulin and analyzed for their effecton cell division.

The SKOV-3 cells expressing EGFP-α-tubulin were synchronized bythymidine block to analyze the effect on cell division of each compound.As shown in the upper part of FIG. 2, each compound was added to theculture medium after the 1st thymidine block. After synchronization bythe second thymidine block, images were taken every 2 minutes for 3 daysusing Leica AF6000 and ×20/0.5 Plan Fluotar objective lens in a phenolred-free RPMI1640 medium supplemented with 5% fetal bovine serum. Thesetime-lapse images were analyzed using ImageJ 1.42a software, and thetime of mitotic arrest was determined by visual judgment. The durationof mitosis was manually determined from the image sequences of at least50 cells.

(3) Results

As shown in FIG. 2, the 4 compounds obtained by screening inducedmitotic arrest in a concentration-dependent manner. The concentrationsrepresent final concentrations. Dose-dependent (EC₅₀=0.3 to 8.0 μg/ml)mitotic arrest was observed for all of the 4 compounds. By contrast,C₇—O-propylated NPD3574 synthesized by propylation at position C₇ didnot arrested mitosis.

Since C₇—O-propylated NPD3574 having the structure of the formula (I)but propylated at position C₇ lost mitotic arrest activity, the OH groupat position C₇ is important for activity. In light of the structures ofthe 4 compounds obtained by screening, the N,N-disubstituted aminomethylgroup at position C₈ was presumed to also serve as a principaldeterminant.

Example 3 Synthesis of Novel Compound

As mentioned above, the compounds represented by the general formula (I)having an OH group at position C₇ and a N,N-disubstituted aminomethylgroup at position C₈, and having TACC3 binding activity were confirmedto have inhibitory activity of cell division. Thus, in order to obtainnovel compounds having TACC3 binding activity and inhibitory activity ofcell division, compounds having the basic structure of the formula (I)were synthesized.

The following 29 compounds were synthesized:

Hereinafter, synthesis methods will be described. Synthesis methods willbe described in detail for two typical compounds RT-007 and RT-011 whichare synthesized using dicoumarin as a starting material and twocompounds RT-002 and RT-027 which are synthesized using the compound ofthe formula (II) (NPD3574) as a starting material. Other compounds canalso be synthesized with the above-mentioned compounds or the like asstarting materials.

(1) Example 3-1 Synthesis of RT-007

The method for synthesizing the starting material dicoumarin follows theprocedure of Dubovic et al. (Non Patent Literature 14).

A 37% aqueous formaldehyde solution (458 μl, 6.21 mmol) and4-(ethylaminomethyl)pyridine (267 mg, 1.96 mmol) were added to astirring solution of dicoumarin (100 mg, 0.327 mmol) in acetonitrile (15ml), and the mixture was heated for 24 hours under reflux. The reactionsolution was cooled to room temperature and diluted with CHCl₃. Theorganic layer was washed with water and saturated saline in this orderand then dried over Na₂SO₄, and the solvent was distilled off. Theresidue was crudely separated by preparative TLC (CHCl₃:MeOH=20:1) andthen further purified by preparative TLC (CHCl₃:MeOH=50:1) to obtainRT-007 (yellow solid, 73.7 mg, yield: 49%).

The NMR data of the compound RT-007 obtained by synthesis is as follows:

¹H NMR (500 MHz, CDCl₃) 6=1.21 (t, J=6.9 Hz, 3H), 2.69 (q, J=6.9 Hz,2H), 3.73 (brs, 2H), 4.16 (s, 2H), 6.28 (s, 1H), 6.74 (d, J=9.2 Hz, 1H),7.20 (d, J=9.2 Hz, 1H), 7.28 (dd, J=4.6, 1.7 Hz, 2H), 7.38 (ddd, J=7.4,7.4, 1.1 Hz, 1H), 7.43 (brd, J=8.6 Hz, 1H), 7.61 (dd, J=7.4, 1.7 Hz,1H), 7.66 (ddd, J=8.6, 7.4, 1.7 Hz, 1H), 7.87 (s, 1H), 8.60 (dd, J=4.6,1.7 Hz, 2H).

¹³C NMR (125 MHz, CDCl₃) 6=11.0, 47.6, 49.9, 57.0, 108.4, 110.5, 112.3,113.8, 116.9, 118.3, 124.0, 124.1, 125.1, 126.5, 128.5, 133.1, 143.3,145.6, 150.1, 150.2, 152.9, 154.2, 159.0, 160.6, 162.8.

HRMS-FAB: m/z [M+H]⁺ calcd for C₂₇H₂₃N₂O₅: 455.1607. found: 455.1589.[M+Na]⁺ calcd for C₂₇H₂₂N₂NaO₅: 477.1426. found: 477.1423.

(2) Example 3-2 Synthesis of RT-011

A 37% aqueous formaldehyde solution (17.7 μl, 240 μmol) and 4-piperidineethanol (9.7 mg, 75.0 μmol) were added at room temperature(approximately 23° C.) to a solution of dicoumarin (9.2 mg, 30.0 μmol)in acetonitrile (3.5 ml). This stirring solution of the reaction mixturewas heated for 24 hours under reflux. The mixed solution was cooled toroom temperature and diluted with CHCl₃. The organic layer was washedwith water and saturated saline in this order and then dried overNa₂SO₄, and the solvent was distilled off. The obtained residue wascrudely separated by preparative TLC (CHCl₃:MeOH=20:1) and then furtherpurified by preparative TLC (CHCl₃:MeOH=10:1) to obtain RT-011 (yellowoily product, 10.2 mg, yield: 76%) as an yellow oil.

The NMR data of the compound RT-011 obtained by synthesis is as follows:

¹H NMR (500 MHz, CDCl₃) 6=1.32 (m, 2H), 1.53 (m, 3H), 1.79 (brd, J=12.6Hz, 2H), 2.24 (brs, 2H), 3.03 (brs, 2H), 3.70 (t, J=6.9 Hz, 2H), 4.04(s, 2H), 6.22 (s, 1H), 6.67 (d, J=9.2 Hz, 1H), 7.15 (d, J=9.2 Hz, 1H),7.36 (brdd, J=7.4, 7.4 Hz, 1H), 7.42 (brd, J=8.6 Hz, 1H), 7.57 (dd,J=7.4, 1.7 Hz, 1H), 7.63 (ddd, J=8.6, 7.4, 1.7 Hz, 1H), 7.82 (s, 1H).

¹³C NMR (125 MHz, CDCl₃) 6=32.0, 39.0, 53.3, 54.3, 60.3, 108.1, 110.2,114.0, 117.0, 118.4, 124.3, 125.0, 126.2, 128.5, 133.0, 143.1, 150.1,152.9, 154.3, 159.0, 160.7, 168.8.

HRMS-FAB: m/z [M+H]⁺ calcd for C₂₆H₂₆NO₆: 448.1760. found: 448.1764.

(3) Example 3-3

RT-002 and RT-027 were synthesized with NPD3574 as a starting materialas follows:

Synthesis of RT-002

An acetic anhydride (4 mL) solution of NPD3574 (6.6 mg, 15 μmol) wasstirred for 10 hours under heating to reflux. The reaction solution wasbrought back to room temperature, and acetic anhydride was thendistilled off under reduced pressure. CHCl₃ was added to the residue.The organic layer was washed with H₂O and saturated saline in this orderand then dried over Na₂SO₄, and the solvent was distilled off. Theresidue was purified by preparative TLC (CHCl₃:MeOH=20:1) to obtainRT-002 (colorless crystal, 5.3 mg, 84%).

(4) Example 3-4 Synthesis of RT-027

m-Chloroperbenzoic acid (77% purity, 3.0 mg, 13.3 μmol) was added to aCH₂Cl₂ (5 mL) solution of NPD3574 (5.0 mg, 11.1 μmol), and the mixturewas stirred at room temperature for 4 hours. Then, the reaction solutionwas diluted with CHCl₃. The organic layer was washed with saturatedaqueous solution of Na₂CO₃ and saturated saline in this order and thendried over Na₂SO₄, and the solvent was distilled off. The residue waspurified by preparative TLC (CHCl₃:MeOH=10:1) to obtain RT-027 (yellowoily product, 3.9 mg, 75%).

Other compounds can also be synthesized in the same way as above by useof amines described in Table 1 below. Table 1 shows amines used in thesynthesis of compounds having inhibitory activity of cell growth asshown in Table 2 below among 30 synthesized compounds.

TABLE 1 Com- pound R¹ Amine RT-002 OAc See Example 3-3 RT-003 NEt₂Diethylamine RT-004 NMe(CH₂CH₂OH) N-methylethanolamine RT-005NH(CH₂CH₂NMe₂) N,N-dimethylethylenediamine RT-006 NEt(CH₂CH₂NMe₂)N,N-dimethyl-N′- ethylethylenediamine RT-007

4- (ethylaminomethyl)pyridine RT-008

Pyrrolidine RT-009

Piperidine RT-010

4-pipecoline RT-011

4-piperidineethanol RT-012

Morpholine RT-014

4-piperidinopiperidine RT-016 N(CH₂CH₂OMe)₂ bis(2-methoxyethyl)amineRT-019

Morpholine RT-027 N⁺O⁻(CH₂CH₂OMe)₂ See Example 3-4 RT-028 N(CH₂CH₂OMe)₂bis(2-methoxyethyl)amine RT-029 N(CH₂CH₂OMe)₂ bis(2-methoxyethyl)amine

Example 4

Assay on mitotic arrest (EC₅₀=50% arrest rate) of SKOV-3 and cell death(IC₅₀=50% survival rate) of lymphoma

The 29 compounds synthesized in Example 3 and NPD3574 used as a startingmaterial in the synthesis of RT-002 and RT-027 were analyzed for theirinhibitory activity of cell growth.

(1) Effect on Mitotic Arrest (EC₅₀=50% Arrest Rate)

The mitotic arrest (EC₅₀=50% arrest rate) was tested by the microscopicanalysis of individual single cells.

EGFP-α-tubulin-expressing SKOV-3 cells synchronized by double thymidineblock in the same way as in Example 2 were treated with each synthesizedcompound and subjected to time-lapse photography for 3 days. At least 50cells were observed. The time of arrest of each cell was calculated, andEC₅₀ was determined therefrom.

(2) Calculation of Cell Death (IC₅₀=50% Survival Rate)

The synthesized compounds were analyzed for their activity against celldeath induction using a mouse lymphoma cell line (see Non PatentLiterature 12), which was a cell line derived from thymic lymphomadeveloped in p53KO mouse.

The number of the lymphoma cells was adjusted to 1.2×10⁵ cells/ml. Thecells were inoculated at a concentration of 50 μl/well to a 96-wellculture dish. A medium containing each RT compound as the novel compoundof the present invention at twice the concentration of 50 μl was furtheradded thereto (final concentration: 0.021 to 13.3 μg/ml). Three daysafter cultivation, the survival rate was calculated using WST-1 reagent(manufactured by F. Hoffmann-La Roche, Ltd.).

(3) Results

Table 2 below describes compounds in order of the strength of inhibitoryeffect on cell growth. The compounds RT-007, RT-016, RT-019, RT-029,RT-011, RT-003, RT-010, RT-006, RT-012, RT-009, RT-008, RT-027, RT-028,RT-014, RT-004, RT-002, RT-005, and NPD3574 were confirmed to haveremarkable inhibitory effects on cell growth.

Particularly, RT-007 was found effective at concentrations as very lowas EC₅₀ of 0.05 μg/ml in the analysis using SKOV-3 and IC₅₀ of 0.02μg/ml in the analysis using lymphoma.

TABLE 2 SKOV3 lymphoma Compound EC₅₀ (μg/ml) IC₅₀ (μg/ml) Remarks RT-007 0.05  0.02 SPL-B RT-016  0.15  0.20 RT-019  0.16  0.12 RT-029  0.17 0.21 RT-011  0.79  2.15 SPL-A RT-003  0.85  5.08 NPD3574  0.85  1.26RT-010  0.94  1.64 RT-006  1.15  3.30 RT-012  1.39  2.05 RT-009  2.6810.13 RT-008  2.67  9.77 RT-027  4.96  8.74 RT-028  6.56  3.28 RT-014 8.76  8.25 RT-004  8.49 13.3< RT-002 13.3<  8.30 Cytotoxic. At 66.7  μg/ml, mitosis did not   start. RT-005 13.3<  5.37 Cytotoxic. At 66.7μg/ml, mitosis did not start. RT-001 66.7< RT-013 66.7< RT-017 66.7<Insoluble RT-018 66.7< Poorly soluble in DMSO RT-020 66.7< InsolubleRT-021 66.7< Insoluble RT-022 66.7< Insoluble. At 66.7 μg/ml, mitosisdid not start. RT-023 66.7< RT-024 66.7< RT-025 66.7< RT-026 66.7<RT-030 66.7<

These results demonstrated that the compounds having the structure ofthe general formula (I) and having the following particular groups asR¹, R², and R³ have a cell growth inhibitory effect:

R¹ represents

OAc, NEt₂, NMe(CH₂CH₂OH), NH(CH₂CH₂NMe₂), NEt(CH₂CH₂NMe₂),N(CH₂CH₂OMe)₂, N⁺O⁻(CH₂CH₂OMe)₂, NMe(CH₂)₃Me, or N(CH₂CH₂Me)₂; R²represents Ac or H; and R³ represents H, Cl, F, or Br.

Since these compounds exhibit a cell growth inhibitory effect, thecompounds used in anticancer agents or the like can be expected toinhibit the growth of tumor cells. Thus, these compounds were analyzedin detail for inhibitory effects on their cell growth, particularly,their effects on cell division.

RT-011 and RT-007 having effects at distinctive concentrations wereselected from 18 compounds determined to have superior effects as tomitotic arrest and cell death among the above-mentioned tested 30 novelcompounds. The selected compounds were designated as SPL-A and SPL-B,respectively, and analyzed in detail. The results will be describedbelow.

Example 5 Effects of SPL-A and SPL-B on Cell Division

Of the observed 18 compounds that had a cell growth inhibitory effect onSKOV-3 and mouse lymphoma, SPL-A and SPL-B were analyzed for theirdose-dependency of mitotic arrest in order to study the effects of theseSPL compounds on cell division.

EGFP-α-tubulin-expressing SKOV-3 cells supplemented with each of SPL-Aand SPL-B at varying concentrations were synchronized by doublethymidine block in the same way as in Example 2. Analysis based onsingle cells was conducted by time-lapse photography. The duration ofmitosis was measured from the image sequences of at least 50 cells.

The concentrations in FIG. 3 represent final concentrations. SPL-Ainduced mitotic arrest at 2.667 μg/ml, whereas SPL-B induced mitoticarrest at 0.107 μg/ml. SPL-B was more highly active than SPL-A againstmitotic arrest. These results are also consistent with the results ofanalysis of mitotic arrest and cell death shown in Table 2.

Example 6 Cell Fate Analysis by Treatment with SPL-A and SPL-B

As shown in FIG. 3, SPL-A and SPL-B induce mitotic arrest in aconcentration-dependent manner. Thus, analysis was made on at whatperiod during the cell cycle mitotic arrest would occur.

(1) Cell Fate Determination by Time-Lapse Photography

The cells used were SKOV-3 cells and OVCAR-3 cells expressingEGFP-α-tubulin.

The OVCAR-3 cells expressing EGFP-α-tubulin were obtained using the sameprocedure as that for the SKOV-3 cells expressing EGFP-α-tubulin. Thecells supplemented with each of SPL-A and SPL-B were synchronized bydouble thymidine block in the same way as in Example 2. Images wereobtained by time-lapse photography and analyzed. The cells of each linewere treated with 13.3 μg/ml SPL-A or 2.7 μg/ml SPL-B. The cell fate wasmanually determined from the image sequence of at least 50 cells withthe time point of nuclear envelope breakdown defined as 0.

(2) Results

The analysis results are shown in FIGS. 4A and 4B. Although mitosis isarrested, and cell growth is inhibited in SKOV-3 and OVCAR-3, the timeof arrest during the cell cycle differs between SKOV-3 and OVCAR-3.

Most of the SKOV-3 cells treated with SPL-A exhibit mitotic slippage.The mitotic slippage occurs by the sustention of cell cycle arrest atthe cell division stage. Some cells underwent death in interphase aftermitotic slippage, and mitotic death. Also, most of the SKOV-3 cellstreated with SPL-B exhibited mitotic slippage, while mitotic death wasobserved in some cells.

On the other hand, most of the OVCAR-3 cells treated with SPL-Aexhibited mitotic death, while death in interphase after mitoticslippage, and mitotic slippage were observed in some cells. Also, mostof the OVCAR-3 cells treated with SPL-B exhibited mitotic death, whilemitotic slippage was observed in some cells. After delayed mitoticarrest, both of the compounds induced mitotic arrest in SKOV-3 orinduced mitotic death in OVCAR-3.

These results are consistent with results of an experiment on cell fatedetermination of SKOV-3 and OVCAR-3 by the suppression of TACC3expression using shRNA. Specifically, the suppression of TACC3expression using shRNA induces mitotic slippage (which occurs by thesustention of cell cycle arrest at the cell division stage) in SKOV-3and mitotic death in OVCAR-3.

The results of analysis using SPL-A and SPL-B are very consistent withthe results of the experiment on the suppression of TACC3 expressionusing shRNA, suggesting that SPL targets TACC3.

Example 7 Change in Abnormal Cell Division Depending on Concentration ofSPL Compound

From the results mentioned above, SPL-A and SPL-B were confirmed toexhibit similar effects on the cell cycle of the same cells. Thus, SPL-Awas added in a concentration-dependent manner, and cells containingspindles with abnormal morphology were analyzed using SKOV-3 cells andOVCAR-3 cells.

(1) Analysis of Abnormal Spindle Morphology in Cell Treated with SPL-A

SPL-A was added at each concentration to the SKOV-3 or OVCAR-3 cells.Twenty four hours after incubation, the cells were fixed andimmunostained using antibodies against cell division-related substancessuch as α-tubulin described below. The percentage of spindle morphologywas determined from at least 50 cells. The data was indicated by a meanof three independent experiments and SD.

The immunostaining was carried out by the following method: the cellswere fixed in 3.7% PFA, methanol, or 10% TCA on a chamber slide, thenpermeabilized with 0.2% Triton-X100 in PBS, and incubated with primaryantibodies. A monoclonal antibody (DM1A) against α-tubulin andmonoclonal antibodies (DQ19 and GTU-88) against γ-tubulin were obtainedfrom Sigma-Aldrich Corp. An anti-centrin 2 antibody (sc-27793) waspurchased from Santa Cruz Biotechnology, Inc. Fluorescent labelled goatanti-mouse IgG (Cappel) and Cy3 labelled goat anti-rabbit IgG(Millipore) antibodies were used as secondary antibodies. DNA wasdetected using 4,6-diamidino-2-phenylindole (DAPI). Images were obtainedusing Leica DM6000B microscope equipped with ×100/1.40-0.70 PlanApoobjective lens and Z-projections.

(2) Results

FIGS. 5A and B show the results of analyzing spindle morphology. Thepercentage of spindle morphology was indicated by the percentage (%) ofnormal, multipolar, disorganized, or monopolar spindles to the number ofmitotic cells. The concentrations represent final concentrations. Ateach concentration, normal, multipolar, disorganized, and monopolar areshown from the left to the right. Normal means the state where spindleswere arranged in a bipolar configuration (normal state). Disorganized,multipolar, or monopolar refers to abnormal spindles.

SPL-A induced multipolar spindles in the cells of both lines. At 1.25μg/ml, 53.3% of SKOV-3 cells and 44.0% of OVCAR-3 cells have multipolarspindles, whereas the percentage of normal spindles drasticallydecreases.

Also, SPL-A induced various abnormal spindles at high concentrations. At5 μg/ml, 56.7% of SKOV-3 cells had disorganized spindles with thehighest percentage. At 2.5 μg/ml, 57.3% of OVCAR-3 cells haddisorganized spindles with the highest percentage. The percentage ofdisorganized spindles decreased with increases in SPL-A concentration,and the percentage of multipolar spindles instead increased. Thepercentage of monopolar spindles increased by high doses of SPL-A.

Although not shown herein, γ-tubulin is localized at the poles of eachspindle, whereas centrin-2 was detected only in those two. Further imageanalysis by time-lapse photography revealed that SPL-A selectivelyinhibits the nucleation of centrosomal microtubules, whereas centromeremicrotubules undergo nucleation and polymerization, resulting in theformation of ectopic spindle poles and multipolar spindles.

The results that SPL-A selectively disrupts the nucleation ofcentrosomal microtubules and induces multipolar spindles reproduce aTACC3 depletion phenotype that suppressed TACC3 expression using shRNA.In addition, OVCAR-3 was more highly sensitive than SKOV-3 to SPL-Atreatment. This is consistent with previous observation in which TOGpdepletion induces more severe spindle malfunction in OVCAR-3 than inSKOV-3.

The SPL treatment reproduces a spindle phenotype induced by TACC3depletion or TOGp depletion. This suggests that SPL-A inhibits theTACC3-TOGp pathway and destabilizes spindle microtubules.

Example 8 Interaction Between TACC3-TOGp Complex and SPL

As mentioned above, the SPL treatment produced results similar to thoseabout the suppression of TACC3 expression using shRNA or the suppressionof TOGp expression, suggesting that SPL acts on spindle microtubules viaa TACC3-TOGp complex. It was thus predicted that SPL might interactdirectly with the TACC3-TOGp complex. Thus, the complex was analyzed forits direct binding to SPL.

(1) Binding of SPL to TACC3 and TOGp

Immobilized SPL on agarose beads was prepared. A cell lysate of SKOV-3was prepared by sonication in a binding buffer (10 mM Tris-HCl (pH 7.5),50 mM KCl, 5 mM MgCl₂, 1 mM EDTA, and protease inhibitor mixtures(manufactured by F. Hoffmann-La Roche, Ltd.)). After removal ofinsoluble materials by centrifugation, the supernatant (500 μg ofproteins) was incubated with 30 μl of the beads at 4° C. for 3 hours.The beads thus incubated were washed four times with a binding buffercontaining 0.025% NP40. The proteins bound with the beads were elutedusing an SDS-PAGE sample buffer, then separated by SDS-PAGE, and thentransferred to nitrocellulose. The blot was incubated with primaryantibodies, subsequently incubated with peroxidase-conjugated secondaryantibodies, then visualized by use of ECL detection system (GEHealthcare Japan Corp.). An antibody against TACC3 or TOGp and as acontrol, antibody against GADPH were used.

The antibody against human TACC3 was produced by the immunization ofrabbits with a peptide corresponding to amino acids 213 to 224 of humanTACC3. The rabbit anti-TOGp antibody was produced against C-terminal 111amino acids of human TOGp fused with GST protein. These antibodies wereused after purification using peptide-bound immunoaffinity columns. Theanti-GADPH antibody (sc-25778) was purchased from Santa CruzBiotechnology, Inc.

(2) Results

In FIG. 6, SPL-A beads, SPL-B beads, and control beads representSPL-A-immobilized agarose beads, SPL-B-immobilized agarose beads, andunimmobilized agarose beads, respectively.

TACC3, TOGp, and GAPDH represent anti-TACC3 antibody, anti-TOGpantibody, and anti-GAPDH antibody treatments, respectively.

As shown in FIG. 6, the specific binding between SPL and TACC3 wasobserved. The signal intensity of TACC3 was stronger in SPL-B than inSPL-A. This is consistent with the higher activity of SPL-B in the cellgrowth inhibition assay. Also, coprecipitates were detected byimmunoblotting using an anti-TOGp protein antibody. TOGp was detectedfrom the SPL beads, indicating the interaction between the TACC3-TOGpcomplex and SPL.

Thus, SPL was shown to inhibit the spindle formation of microtubules viadirect binding to the TACC3-TOGp complex to induce abnormal spindlemorphology and cell division inhibition. These results indicate that SPLtargets not only TACC3 but TOGp. Although only the results about SPL areshown herein, compounds having the common structure of the formula (I)is presumed to induce cell growth inhibition under a similar mechanism.

Example 9 In Vivo Antitumor Effect of SPL-B

Next, the ability of SPL to act as an anticancer agent is shown using anin vivo experimental system.

(1) Method

All animals and methods were approved by the Japanese Foundation forCancer Research (JFCR, Japan) Cancer Institute Animal Committee. Forxenograft tumor models, 1×10⁷ cells of ovary cancer cells, SKOV-3 cells,were subcutaneously injected to both flanks of 9 NOD/SCID mice (CharlesRiver Laboratories Japan, Inc.). When their tumor volumes reachedapproximately 200 mm³, 50 μl of SPL-B dissolved in DMSO was orallyadministered to each mouse every 2 days. The tumor volumes were measuredevery 2 days. DMSO was administered as a control by the same procedureas above. Two or 3 mice were analyzed in each group. In this context,the tumor volumes were determined by measuring the length (L) and width(W) of each tumor using calipers and calculating the tumor volume (tumorvolume=L×W×W/2).

Thirteen days after the treatment, the mice were dissected to collecttissues, which were then fixed overnight in 10% buffered formalin (WakoPure Chemicals Industries, Ltd.,) and embedded in paraffin to prepare1-, 4- or 6-μm sections. The sections were immunostained with anti-p53,anti-p21, anti-H3K9me3, and anti-PCNA antibodies. The percentage ofpositive cells in at least 10 regions arbitrarily selected in twosections was measured. The data was indicated by a mean and SD.

(2) Results

The start date of SPL-B administration is defined as day 1. The tumorvolume at the start day of SPL-B administration is defined as 1.

As shown in FIG. 7, SPL-B inhibited tumor growth in a dose-dependentmanner in vivo.

In addition, SPL-B was orally or intraperitoneally administered at adose of 20 mg/kg. Six hours after administration, its concentrations inserum were measured. As a result, the orally administered SPL-B showed aserum concentration of 270 ng/ml, whereas the intraperitoneallyadministered SPL-B showed a serum concentration of 90 ng/ml. Thus, SPL-Bexhibits higher concentrations in blood by oral administration and hasvarious clinical applicability for treatment.

As shown in FIG. 8, the SPL-B treatment enhances the expression of p53,p21, and K9 trimethyl histone H3 (H3K9me3) which induce cell death andsuppresses the expression of PCNA which induces cell growth. Theseresults indicate that the anticancer effect of SPL-B is attributed tothe activation of the p53-p21 pathway.

Example 10 Effect of SPL-B on Various Cell Lines

Provided that SPL acts on spindle formation and mitotic apparatuscontrol via the TACC3-TOGp complex, SPL is considered to have a cellgrowth inhibitory effect on a wide range of cancer types. Thus, theeffect of SPL was assayed using various cancer cell lines.

(1) Method

SPL-B was allowed to interact on cell lines of human cancer cell linepanel (Non Patent Literature 14) and analyzed for the effects of SPL oncell division.

The cells were treated with SPL-B at a concentration of 0.5 μg/ml for 24hours, then stained with propidium iodide (PI), and analyzed by flowcytometry. FIG. 9 shows the changes in G2/M-phase cells by the SPL-Btreatment.

(2) Results

Among 39 cell lines used in the analysis, over half thereof (21 celllines) exhibited increase in the percentage of G2/M-phase cells,demonstrating that SPL has the effect of arresting cell division.Particularly, the ovary cancer-derived cell lines SKOV-3 and OVCAR-3,the colon cancer-derived cell lines HCT15 and KM12, the glioma-derivedcell lines SNB-75 and SNB-78, the prostate cancer-derived cell linesPC-3 and DU145, the kidney cancer-derived cell line ACHN, and themelanoma-derived cell line LOX-IMVI exhibited marked increase in thepercentage of G2/M-phase cells, indicating the high celldivision-arresting effect by SPL.

Example 11 Selective Growth Inhibitory Effect of SPL on Cancer Cell

Next, SPL, unlike other mitotic poisons or kinase inhibitors, is shownto cause no growth inhibition in normal cells, though being effectivefor the growth inhibiting of cancer cells.

(1) Method

(A) Two cancer cell lines SKOV-3 and OVCAR-3 and three normal cell lineshTERT-RPE, MCF 10A, and 184B5 were treated with SPL-A at concentrationsshown in the abscissa of FIG. 10A for 24 hours, then stained withpropidium iodide (PI), and analyzed by flow cytometry. FIG. 10A showsthe rate of changes in G2/M-phase cells by the SPL-B treatment.

(B) Agents known to act on spindle microtubules to inhibit tumor growthwere analyzed for their effects.

The same two cancer cell lines and three normal cell lines as in (A)were treated with each of paclitaxel, vincristine, and vinblastineacting on microtubule formation, S-trityl-L-cysteine (STLC) and SB743921acting on proteins involved in mitosis, an Aurora kinase inhibitorVX680, and a polo-like kinase (PLK) inhibitor BI2536 at concentrationsshown in the abscissa of FIG. 10B for 24 hours, then stained withpropidium iodide (PI), and analyzed by flow cytometry. FIG. 10B showsthe rate of changes in G2/M-phase cells by the treatment with eachagent.

(2) Results

As shown in FIG. 10A, SPL-A increased the percentage of G2/M-phase cellsin a concentration-dependent manner in the two cancer cell lines andthus has the effect of arresting cell division. However, even at highconcentrations SPL acts no inhibitory effect on cell division of the 3normal cell lines. Thus, SPL-A was shown to selectively inhibit oncancer cells.

By contrast, all of other compounds or agents known to act onmicrotubular polymerization/depolymerization or spindle formationassociated therewith inhibited the division of all the cell lines in aconcentration-dependent manner and thus lack a selective effect on tumorcells as seen in SPL (FIG. 10B).

Thus, it was shown that the effect of inhibiting the division of cancercells, but not acting on normal cells is unique to SPL, whereas othermitotic poisons or kinase inhibitors, etc. lack such properties. Sincethe compound of the present invention specifically acts on cancer cellsbut does not act on normal cells, the compound of the present inventionused as an anticancer agent is likely to cause no serious adversereaction.

Example 12 Effect Brought about by Combined Use of SPL and AdditionalAnticancer Agent

Next, effects brought about by combined use with an anticancer agentknown in the art were studied. If the compound of the present inventionused in combination with an additional agent has an enhancing effect,the dose of each agent can be decreased, resulting in reduced adversereactions.

p53-knockout cells derived from human colon cancer cells HCT-116 werecultured after combined administration of paclitaxel and SPL-B. Threedays after cultivation, the survival rate of the cells was measured byWST assay. The results are shown in FIG. 11A.

FIG. 11A shows the results of measuring, by WST assay, the cell growthof the p53-knockout HCT-116 cells cultured with combined addition ofSPL-B at concentrations of 0, 150, and 300 μM and paclitaxel in aconcentration range of 0 to 32 nM. IC₅₀ of paclitaxel was 8.339 nM inthe absence of SPL-B, 7.697 nM in the case of adding SPL-B at 150 μM,and 7.537 nM in the case of adding SPL-B at 300 μM.

As shown in FIG. 11B, SPL-B alone is rarely effective for cell growtheven when added at 300 μM to the medium, whereas the combined usethereof with paclitaxel can enhance the cell growth inhibitory effect.Paclitaxel, a microtubular depolymerization inhibitor, inhibits celldivision as with SPL-B. SPL-B, however, differs in the mechanism ofaction from paclitaxel and is therefore considered to have an enhancingeffect. As shown above, the compound of the present invention is orallyadministrable and acts on spindles via TACC3 or a TACC3-TOGp complexwithout acting on microtubules themselves. Thus, the compound of thepresent invention does not inhibit the functions of microtubules even innon-dividing cells, unlike vinca alkaloids currently used as anticanceragents targeting microtubules. Accordingly, the compound of the presentinvention is free from serious adverse reactions such as peripheralneuropathy.

INDUSTRIAL APPLICABILITY

The present invention provides a novel anticancer agent targeting theTACC3 protein. The compound of the present invention acts on individualsthrough oral administration and inhibits the functions of spindles atlow concentrations. The compound of the present invention can thereforebe used as an anticancer agent with few adverse reactions.

1. A compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of a cancer targeting at TACC3 and/or a TACC3-TOGp complex:

wherein R¹ represents

OAc, NEt₂, NMe(CH₂CH₂OH), NH(CH₂CH₂NMe₂), NEt(CH₂CH₂NMe₂), N(CH₂CH₂OMe)₂, N⁺O⁻(CH₂CH₂OMe)₂, NMe(CH₂)₃Me, or N(CH₂CH₂Me)₂; R² represents Ac or H; and R³ represents H, Cl, F, or Br.
 2. The compound or the pharmaceutically acceptable salt thereof for use in the treatment of a cancer, targeting at TACC3 and/or a TACC3-TOGp complex according to claim 1, wherein the cancer targeting TACC3 is colon cancer, ovary cancer, uterine cancer, breast cancer, esophagus cancer, lymphoma, glioma, prostate cancer, kidney cancer, or melanoma.
 3. A method for producing an anticancer agent composition, comprising mixing a compound or a pharmaceutically acceptable salt thereof according to claim 1 with a pharmaceutically acceptable excipient.
 4. The compound or the pharmaceutically acceptable salt thereof for use in the treatment of a cancer, targeting at TACC3 and/or a TACC3-TOGp complex according to claim 1, wherein the compound or the pharmaceutically acceptable salt thereof is selected from the group consisting of: 